Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
Mammals are required to defend themselves against a multitude of pathogens including viruses, bacteria, fungi and parasites, as well as non-pathogenic insults such as tumours and toxic, or otherwise harmful, agents. In response, effector mechanisms have evolved which are capable of mounting a defense against such antigens. These mechanisms are mediated by soluble molecules and/or by cells.
In the context of these effector mechanisms, inflammation is a complex multifaceted process in response to disease or injury which is regulated by the release of a series of cytokines (Alexander et al, 2001, J Endotoxin Res 7:167-202). These cytokines are classified in general terms as pro- or anti-inflammatory cytokines and the critical balance between release and activity of cytokines with opposing actions regulates the inflammatory response to prevent it from becoming overt or understated.
If the inflammatory response continues unchecked and is overt then the host may suffer associated tissue damage and in severe cases this may present as septic shock and multi-organ failure can occur (Ulevitch et al., 1999, Curr Opin Immunol 11:19-22). Conversely, a poor or understated inflammatory response may mean uncontrolled infection resulting in chronic illness and host damage. Regulation of the inflammatory response is important at both the systemic level and the local level.
The discovery of the detailed processes of inflammation has revealed a close relationship between inflammation and the immune response. There are five basic indicators of inflammation, these being redness (rubor), swelling (tumour), heat (calor), pain (dolor) and deranged function (functio laesa). These indicators occur due to extravasation of plasma and infiltration of leukocytes into the site of inflammation. Consistent with these indicators, the main characteristics of the inflammatory response are therefore:    (i) vasodilation—widening of the blood vessels to increase the blood flow to the infected area;    (ii) increased vascular permeability—this allows diffusible components to enter the site;    (iii) cellular infiltration—this being the directed movement of inflammatory cells through the walls of blood vessels into the site of injury;    (iv) changes in biosynthetic, metabolic and catabolic profiles of many organs; and    (v) activation of cells of the immune system as well as of complex enzymatic systems of blood plasma.
The degree to which these characteristics occur is generally proportional to the severity of the injury and/or the extent of infection.
The inflammatory response can be broadly categorised into several phases. The earliest, gross event of an inflammatory response is temporary vasoconstriction, i.e. narrowing of blood vessels caused by contraction of smooth muscle in the vessel walls, which can be seen as blanching (whitening) of the skin. This is followed by several phases that occur over minutes, hours and days later, as follows:    (i) The acute vascular response follows within seconds of a tissue insult and lasts for some minutes. It is characterised by vasodilation and increased capillary permeability due to alterations in the vascular endothelium, leading to increased blood flow (hyperaemia) that causes redness (erythema) and the entry of fluid into the tissues (oedema).    (ii) If there has been sufficient damage to the tissues, or if infection has occurred, the acute cellular response takes place over the next few hours. The hallmark of this phase is the appearance of granulocytes, particularly neutrophils, in the tissue. These cells first attach themselves to the endothelial cells within the blood vessels (margination) and then cross into the surrounding tissue (diapedesis). If the vessel is damaged, fibrinogen and fibronectin are deposited at the site of injury, platelets aggregate and become activated and clot formation occurs.    (iii) If damage is sufficiently severe, a chronic cellular response may follow over the next few days. A characteristic of this phase of inflammation is the appearance of a mononuclear cell infiltrate composed of macrophages and lymphocytes. The macrophages are involved in microbial killing, in clearing up cellular and tissue debris, and are also thought to play a significant role in remodelling tissue.    (iv) Over the next few weeks, resolution may occur wherein normal tissue architecture is restored. Blood clots are removed by fibrinolysis. If it is not possible to return the tissue to its original form, scarring may occur from in-filling with fibroblasts, collagen, and new endothelial cells. Generally, by this time any infection will have been overcome, although this is not always the case and may result in further immunological responses, such as granuloma formation.
Inflammation is often considered in terms of acute inflammation that includes all the events of the acute vascular and acute cellular response (1 and 2 above), and chronic inflammation that includes the events during the chronic cellular response and resolution or scarring (3 and 4).
It should be understood, however, that in addition to the occurrence of inflammatory responses in a localised fashion in tissue which is damaged, infected or subject to an autoimmune response inflammatory responses may also occur systemically, such as in the case with sepsis.
Accordingly, in light of the wide-ranging impact of inflammatory responses, there is an ongoing need to elucidate the complex mechanisms by which they function. By identifying these mechanisms there is thereby provided scope for developing means of appropriately modulating inflammatory responses.
Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of follicle stimulating hormone release from pituitary cell culture. In addition to their effects on follicle stimulating hormone synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin.
Activins, composed of two β-subunits, βA, βB, βC and/or βE are members of the transforming growth factor (TGF)-β superfamily [Vale et al., 1990, Handbook of Experimental Physiology, Vol. 95, Eds. Sporn & Roberts, Springer-Verlag, Berlin pp 211-248]. Multimeric protein forms of activin include the homodimeric forms (Activin A-βAβA, Activin B-βBβB, Activin C-βCβC, and Activin E-βEβE) and the heterodimeric forms (for example, Activin AB-βAβB, Activin AC-βAβC, or Activin AE-βAβE). The activins are multifunctional proteins. For example, Activin A, although originally identified as a regulator of follicle stimulating hormone release, is now known to exhibit the pleiotropic range of functional activities which are characteristic of most cytokines. Activins, like their related proteins, inhibins (which consist of a dimer of a structurally related but dissimilar α subunit and an activin β subunit) can bind to activin type II receptors. However, only activins are able to recruit type I receptors to form an active complex, triggering intracellular Smad signalling pathways and thereby influencing cellular function at the transcriptional level. At present, activin A, AB and B have been shown to demonstrate typical receptor-mediated agonist activity. Activin B has been reported to display less biological activity than activin A [Nakamura et al., Journal of Biological Chemistry, 267, 16385-16389, 1992]. This may be associated with variation in the availability of specific type I receptors, differentially recruited by activin A and B [Tsuchida et al., 2004 Molecular and Cellular Endocrinology 220, 50-65].
Follistatin functions as a biological regulator of activin. In fact, it was originally identified by its ability to suppress the secretion of follicle stimulating hormone, subsequently shown to be due to its property as an activin binding protein. Follistatin is a monomeric protein which binds to activin with high affinity and is believed to thereafter lead to lysosomal degradation of the complexed activin. Follistatin comprises a number of post-translational and glycosylation variants. However, the two major isoforms are the full length follistatin 315, which is believed to be the predominant circulating isoform, and the 288 isoform, which has a strong affinity for heparin sulphate proteoglycans and is largely a cell membrane-associated isoform (Phillips and de Kretser, 1998, Frontiers in Neuroendocrinology 19:287-322).
Activin affects the growth and differentiation of many cell types, stimulates the secretion of follicle-stimulating hormone from the pituitary gland and inhibits growth hormone, prolactin, and adrenocorticotropin release [Billestrup et al., Molecular Endocrinology 1990 4:356-362; Kitaoka et al., Biochemical and Biophysical Research Communications 1988 157:48-54; Vale et al., Nature 1986, 321:776-779]. Activin A was first characterized for its ability to stimulate follicle stimulating hormone (FSH) from the pituitary, a capacity shared by activin B [Nakamura et al., 1992, supra; Van Dijk et al., 1995, Annals of the New York Academy of Science 762, 319-330]. However, activin A is now known to have many more properties besides this initial function for which it was first isolated. Both activin A and B participate in foetal development, with their respective mouse knockouts [Vassali et al., 1994, Genes and Development, 8:414-427] presenting distinct phenotypic anomalies. Knockouts of activin A exhibit neonatal lethal phenotypic defects [Vassalli et al., 1994, supra; Matzuk et al., 1995, Nature 374: 354-356] but substitution of the βA gene with βB provides partial rescue of this phenotype [Brown et al., 2000, Nature Genetics, 25:453-457], suggesting some overlap in the activities of activin A and B. In contrast to these observations there is evidence that activin B may have specific roles in processes such as embryonic mesoderm induction [Thomsen et al., 1990, Cell 63:485-493] and mammary gland development [Robinson et al., 1997, Development 124:2701-2708]. Of particular interest is that activin B is presumed to be the activin of relevance in intrapituitary regulation of FSH, as shown by neutralization studies [Corrigan et al., 1991, Endocrinology 128:1682-1684]. Additionally, distinct differences in expression patterns of activin A and B are evident during tissue repair [Hübner et al., 1996, Developmental Biology 173:490-498] and in association with models of liver fibrosis [De Bleser et al., 1997, Hepatology, 26:905-912]. Such evidence suggests that activin A and B play different roles in a range of biological and pathological processes.
Follistatin specifically binds several members of the TGF-β superfamily, but has by far the highest affinity of binding to activin. As a result, circulating follistatin 315 neutralizes activin activity by preventing the interaction of the cytokine with its type II receptors [de Winter et al., Molecular and Cellular Endocrinology 1996 116:105-114] and, furthermore, cell surface-bound follistatin 288 facilitates the lysosomal degradation of activin [Hashimoto et al., Journal of Biological Chemistry 1997 272:13835-13842]. Both follistatin and activin mRNAs show a broad tissue distribution [Meunier et al., PNAS 1988 85:247-251; Michel et al., Biochemical and Biophysical Research Communications 1990 173:401-407; Schneider et al., European Journal of Endocrinology 2000 142:537-544]. Follistatin and activin are detectable in serum [Demura et al., Journal of Clinical Endocrinology and Metabolism 1993 76:1080-1082; Demura et al., Biochemical and Biophysical Research Communications 1992 185:1148-1154; Gilfillan et al., Clinical Endocrinology 1994 41:453-461; Khoury et al., Journal of Clinical Endocrinology and Metabolism 1995 80:1361-1368; Knight et al., Journal of Endocrinology 1996 148:267-279; McFarlane et al., European Journal of Endocrinology 1996 134:481-489; Sakai et al., Biochemical and Biophysical Research Communications 1992 188:921-926; Sakamoto et al., European Journal of Endocrinology 1996 135:345-351; Tilbrook et al., Journal of Endocrinology 1996 149:55-63; Wakatsuki et al., Journal of Clinical Endocrinology and Metabolism 1996 81:630-634], and their concentrations in serum increase with age [Wakatsuki et al. 1996, supra; Loria et al., European Journal of Endocrinology 1998 139:487-492]. At present, however, the precise sources of follistatin and activin in serum are unknown. Current data suggest that tissue-specific balances of follistatin and activin govern the growth and differentiation of responsive cell types in an autocrine/paracrine manner [Michel et al., Acta Endocrinologica 1993 129:525-531; Phillips, Trends in Endocrinology and Metabolism 2001 12:94-96].
An emerging role for activin and follistatin in the body's innate immune response has been documented. For instance, activin and follistatin are secreted by various cell types in response to inflammatory compounds in vitro [Hübner et al., Experimental Cell Research 1996 228:106-113; Jones et al., Endocrinology 2000 141:1905-1908; Keelan et al., Placenta 2000 21:38-43; Michel et al., Endocrinology 1996 137:4925-4934; Phillips et al., Journal of Endocrinology 1998 156:77-82; Yu et al., Immunology 1996 88:368-374; Erämaa et al., Journal of Experimental Medicine 1992 176:1449-1452; Shao et al., Cytokine 1998 10:227-235; Mohan et al., European Journal of Endocrinology 2001 145:505-511]. Moreover, in some examples of inflammatory processes such as wound healing, inflammatory bowel disease and rheumatoid arthritis, increased activin and/or follistatin expression has been noted [Hübner et al., Laboratory Investigation 1997 77:311-318; Hübner et al., 1996, supra; Yu et al., Clinical and Experimental Immunology 1998 112:126-132]. However, since these very early and preliminary findings, the role of activin and follistatin in the context of inflammation, per se, has not been further elucidated, either in the context of their precise activities or in the context of the scope of the inflammatory conditions in which they function. In light of the extreme diversity in terms of the nature and extent of inflammatory responses which can occur, and the extremely pleiotropic activities of cytokines such as the various forms of activin, it is not surprising that the preliminary findings of the mid to late 1990's have not progressed to more substantial theories. In particular, activin A, activin B and follistatin are expressed by a wide variety of cell types and most organs in the body in response to a wide range of stimuli. Accordingly, their role in the context of inflammation cannot be predicted and is therefore far from clear.
In work leading up to the present invention it has been surprisingly determined that activins A functions as a crucial component of the cytokine cascade which regulates the inflammatory response. Specifically, activin A initiates the release, in vivo, of the pro-inflammatory cytokines and can, in fact, modulate the levels of pro-inflammatory cytokines which are released subsequently to an appropriate stimulus. Accordingly, although it has previously been observed that activin A levels are modulated during the onset and progress of an inflammatory response, until the advent of the present invention there had been no progress made in elucidating the precise role of this molecule in the context of inflammation.
It has still further been surprisingly determined that activin B levels are even more dramatically modulated in the context of an inflammatory response than are activin A levels. This is particularly surprising in light of what has been known to date in relation to the distinct roles of activins A and B. Still further, whereas immunoassays directed to the measurement of activin A have been available for use for some time, analysis of activin B has been inhibited by the absence of a specific immunoassay for this particular activin species. A very limited data set is available which suggests that circulating activin B levels alter during pregnancy or with ovarian function [Petraglia et al., 1993, Endocrine Journal 1:323-327; Woodruff et al., 1997, Journal of Endocrinology 152:167-174; Vihko et al., 1998, Human Reproduction 13:841-846; Vihko et al., 2003, Acta Obstetricia et Gynecologica Scandinavica, 80:570-574]. Kobayashi et al. (2000) [Biol. Pharm. Bull. 23(6):755-757] demonstrated that an increase in activin-βB mRNA is associated with liver regeneration and the development of fibrosis, although the authors do not postulate whether this is linked with changes to levels of activin AB, Activin B or inhibin. A study by Rosendahl et al. 2001 [Am J Respir Cell Mol Biol 25:60-68] examined a mouse model of allergen-induced airway challenge in the lung and focussed on examining associated changes in expression and distribution of TGF-β superfamily and TGF-β/activin receptors. This group reported that induced airway allergens produced only a very modest elevation of activin βB mRNA expression over control levels. Histological examinations did not provide any information on mature activin dimer protein synthesis or distribution (either activin A or B) nor was there any evidence provided that the modest increase in activin-βB mRNA levels was not, in fact, linked to changes in inhibin levels. Accordingly, the determination that activin B levels are in fact dramatically increased during inflammation relative to activin A levels is extremely unexpected in light of the very limited information which was available about the functioning of both the activin A and activin B molecules.
The findings of the present invention have now facilitated the development of methodology directed to modulating the inflammatory response by regulating the levels of functionally active activin A and activin B and, therefore, pro-inflammatory cytokine release. Accordingly, there are now provided both methods for the therapeutic or prophylactic treatment of conditions characterised by an unwanted or inappropriate inflammatory response and means for screening for regulators of pro-inflammatory cytokine release such as activin A and activin B mimetics, agonists or antagonists.